Transgenic mice (Tg) overexpressing murine apoA-II have elevated total cholesterol and HDL-C levels but increased rather than decreased atherosclerosis. To evaluate the effectiveness of apoA-II transgenic HDL in reverse cholesterol transport (RCT) we have compared cholesterol efflux with control and apoA-II Tg HDL. Cultured human fibroblasts were radiolabeled with [14C]FC-PC liposomes, [14C] and [3H]oleate, and [14C]acetate to label cholesterol in the plasma membrane (PM), the cholesterol ester storage pool and the smooth endoplasmic reticulum (SER), the site of de novo synthesis of free cholesterol (FC), respectively. There were no statistically significant differences in PM cholesterol efflux rates between control and apoA-II TG HDL, however efflux of newly synthesized cholesterol by apoA-II Tg HDL was reduced by 40-45% (P<0.01) when compared to control HDL. Control HDL caused a concentration-dependent and saturable 50% reduction in the rate of [14C]oleate incorporation into cholesteryl[14C]oleate compared to a more efficient 70-83% reduction in the presence of apoA-II Tg HDL (P<0.01). Efflux of cholesterol from cells with control and apoA-II Tg HDL revealed two distinct sites of cellular cholesterol metabolism that were differentially regulated by apoA-II Tg HDL, the SER pool (decreased efflux) and the cholesteryl ester cycle (more efficient reduction of esterification) Murine apoA-II HDL is dysfunctional in regulating cholesterol metabolism providing a potential mechanism that may in part account for the increased atherosclerosis in the murine apoA-II transgenic mouse model system.